The ultimate goal of this proposed research is the development of a live vaccine for the prevention of Salmonella infection of and subsequent shedding from poultry, in order to reduce the reservoir of infection for man. Single temperature-sensitive mutants of a chicken-virulent strain of Salmonella enteritidis will be isolated, using standard methods of mutagenesis and enrichment, and characterized with respect to phenotype at the body temperature of chickens, reversion frequency, linkage of the ts mutations to selectable chromosomal markers. The single ts strains will also be evaluated for immunogenic potential in a model of oral immunization and challenge in chickens. Strains of S. enteritidis containing multiple mutations of identical temperature-sensitive phenotype, utilizing the linkage of the ts lesions to the selectable markers, will then be constructed. Such strains will contain all the surface antigens in their natural conformation, but will be incapable of sustained replication in the vaccinee. Their reversion frequency will be reduced to negligible levels (-10-21) ensuring their safety, both for the vaccinee and susceptible contacts of the vaccinee. Oral administration of the vaccine strains should induce the formation of specific mucosal antibodies, preventing colonization and, therefore, disease and sustained shedding. The three ts mutations, once characterized and present in a "master" strain, would be suitable for incorporation into other appropriate S. enteritidis strains for the construction of vaccines to prevent Salmonella disease in a variety of animal and human systems.